Anglicheau Dany, Sanquer Sylvia, Loriot Marie Anne, Beaune Philippe, Thervet Eric
INSERM U490, Centre Universitaire des Saints Pères, 45 rue des Saints Pères, F-75270 Paris Cedex 06, France.
J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Jun 25;773(2):119-27. doi: 10.1016/s1570-0232(02)00130-7.
Thiopurine methyltransferase (TPMT) is a cytosolic enzyme involved in the metabolism of thiopurine drugs. A genetic polymorphism is responsible for large inter-individual differences observed in TPMT activity. We report a new HPLC technique, which avoids an extraction step and the use of radioactive reagents, based on the conversion of 6-mercaptopurine (6-MP) to 6-methylmercaptopurine (6-MMP) using S-adenosyl-L-methionine (SAM) as methyl donor in red blood cell lysates (RBC). Intra- and inter-assay variation, within-day, within-run, between-day, and between-run variations showed high precision. The formation of 6-MMP was linear with respect to the lysate concentration and time. In a blinded assay of 61 samples, the results of HPLC method correlated with those of the radiochemical method (r2=0.82, P<0.0001). Using a cut-off point of 8.5 nmol/h/ml packed RBC, positive predictive value of HPLC was 100% for heterozygous patients. Because of the absence of extraction step, this new HPLC technique of TPMT activity determination reduces analysis variation and is time-saving. This rapid, sensitive, and reproducible method is suitable for routine monitoring of TPMT activity and for fundamental studies.
硫嘌呤甲基转移酶（TPMT）是一种参与硫嘌呤类药物代谢的胞质酶。基因多态性导致了TPMT活性存在较大的个体间差异。我们报告了一种新的高效液相色谱（HPLC）技术，该技术基于在红细胞裂解液（RBC）中以S-腺苷-L-甲硫氨酸（SAM）作为甲基供体，将6-巯基嘌呤（6-MP）转化为6-甲基巯基嘌呤（6-MMP），从而避免了萃取步骤和放射性试剂的使用。批内和批间变异、日内、批内、日间和批间变异均显示出高精度。6-MMP的形成与裂解液浓度和时间呈线性关系。在对61个样本的盲法检测中，HPLC方法的结果与放射化学方法的结果相关（r2 = 0.82，P < 0.0001）。使用8.5 nmol/h/ml压积红细胞的截断点，HPLC对杂合子患者的阳性预测值为100%。由于无需萃取步骤，这种测定TPMT活性的新HPLC技术减少了分析变异且节省时间。这种快速、灵敏且可重复的方法适用于TPMT活性的常规监测和基础研究。
